TonB dependent transport processes through Escherichia coli FepA.

Abstract

One of the previously proposed mechanisms of TonB is called the "shuttle hypothesis" which states that TonB shuttles from the inner membrane to the outer membrane. The data presented here demonstrates that TonB does not need to leave the inner membrane to accomplish its function, thereby presenting evidence against this hypothesis.Gram negative bacteria use elaborate transport systems to acquire nutrients from their environment. One type of system is the ligand gated porin (LGP) transport system. All LGP's are TonB dependent and require energy in the form of proton motive force (PMF) to transport their substrates. LGP's are beta-barrels with globular N-domains that reside in the barrel blocking the passage of molecules. Therefore in order for transport to occur the N-domain must change in conformation. This change in conformation is dependent on TonB, PMF and the presence of Ferric Enterobactin (FeEnt).The tail protein of H8 that binds to FepA was compared to the tail proteins of the other bacteriophage T5 and BF23 that are also in the Siphovirida family. Relative to these other tail proteins H8's tail protein contains acidic and aromatic residues that are unique to it and are probably involved in the binding of the phage to FepA. Other than these residues this protein is similar to T5's tail protein suggesting a similar poreforming mechanism through the cell envelope. Of further interest is a region on the H8 tail forming protein that contains a TonB box sequence. This suggests a link between this sequence and TonB dependent processes.H8 is a bacteriophage that was recently discovered to be FepA dependent for its infection of E. coli and the binding of H8 to E. coli is inhibited (50% inhibition concentration, 98 nM) by FeEnt the native siderophore of FepA. H8 did not require the other ferric catecholate receptors (Fui, Cir, or Iron) for infection. H8 has morphology and genomic structure of similarity to bacteriophage T5, a member of the family Siphoviridae. The DNA sequence of H8 showed that H8 has a 104.4 kb genome with a total of 143 open reading frames (ORFS). Of the 143 ORFS 120 of them are homologous to ORFS found in T5.TonB is a 239 residue protein anchored in the inner membrane, spanning into the periplasm of E. coli. It is required by all LGP's for their transport of siderophores and their susceptibility to group B bacteriocins and certain bacteriophage. The mechanism by which TonB accomplishes this is not yet understood. Several fusion proteins were constructed consisting of the green fluorescent protein (GFP) from Aequaria victoria either attached to the C-terminus or the N-terminus of TonB. All of the fusion proteins when expressed in E. coli were active with respect to the transport of FeEnt and the susceptibility to colicin B and D.One of E. coli's LGP transport systems is the FepA system. The FepA system is used for the acquisition of iron. Located in the outer membrane of E. coli it binds and transports ferric enterobactin (FeEnt) a siderophore. In order for transport to occur through FepA the globular N-domain needs to either leave the barrel completely or structurally change inside the barrel to allow for transport through the barrel. To study the transport mechanism of FepA, the susceptibility of 25 cysteine mutants at different locations in FepA to alkylation by fluorecein maleimide (FM) was studied in live cells. The reactivity of those cysteine residues changed in response to the addition of FeEnt. This reactivity was also dependent on the presence or absence of TonB and energy. Of particular note is the reactivity of a residue located deep inside the barrel and the dependence of this labeling on the presence of TonB and energy. Also demonstrated is the functional complementation between the N-terminus and C-terminal barrel expressed separately. The data collected offer support for the ball-and-chain theory for LGP transport